Oncogenic CARD11 mutations and autonomous BCR signaling as functionally equivalent alternative drivers in ABC-DLBCL
Diffuse large B-cell lymphoma of activated B-cell type (ABC-DLBCL) is characterized by chronic signaling of the B-cell receptor (BCR) pathway. We have recently identified autonomous, i.e. antigen-independent signaling as the cause of BCR pathway activation in the majority of ABC-DLBCL cases (Eken et al., J Exp Med 2024). As a pure immunological driver, autonomous BCR signaling cannot be recognized by sequence analysis nor be readily added to proposed genetic DLBCL subclassifications. Besides autonomous BCR signaling, recurrent activating mutations of various members of the BCR signaling cascade contribute to the ABC phenotype. Intriguingly, lack of autonomous BCR signaling is associated with CARD11 L251P mutation in the OCI-Ly3 ABC-DLBCL cell line.
We therefore investigated the function of recurrent CARD11 mutations in CARD11 wild-type ABC-DLBCL cell line TMD8. TMD8 cells express a BCR with autonomous signaling and are highly sensitive to the BTK inhibitor acalabrutinib (IC50 = 0.005 nM). TMD8 cells were gene edited by CRISPR/cas9 homology-directed repair to express the presumed oncogenic CARD11 coiled-coil region mutations L251P, D230N, K215N, and R337Q. BCR pathway activation was analyzed by BTK-inhibition and BCR knock-out (KO). In a reciprocal driver-targeting approach, OCI-Ly3 was transduced with the TMD8 BCR and a CRISPR/cas9-resistant but otherwise wild-type CARD11 allele.
Introduction of a hemizygous CARD11L251P/- allele converted TMD8 cells to full acalabrutinib resistance (IC50 >62 μM). Hemizygous and homozygous R337Q mutations had negligible effects (CARD11R337Q/-: IC50 = 0.003 nM; CARD11R337Q/R337Q: 0.007 nM). Homozygous K215N and D230N mutations induced partial resistance to acalabrutinib (CARD11K215N/K251N clones: IC50 = 0.25-0.42 nM; CARD11D230N/D230N: IC50 = 0.127 nM). In accordance with the observed sensitivities of CARD11-edited TMD8 clones to BTK inhibition, only CARD11L251P/- clones were able to survive KO of their autonomously signaling BCR by CRISPR/cas9-mediated KO. Furthermore, competitive co-culture of TMD8 CARD11L251P/- clones with mock gene-edited control clones failed to show any growth advantages. CRISPR/cas9-mediated knock-out of the CARD11L251P alleles increased acalabrutinib sensitivity from an IC50 >96 μM in CARD11L251P/L251P/tgCARD11WT OCI-Ly3 cells to 0.002-7.2 μM in CARD11ko/ko/tgCARD11WT cells.
In conclusion, we have developed a system to quantify the effects of defined mechanisms of BCR pathway activation in ABC-DLBCL. Our reciprocally controlled results demonstrate that the activating CARD11 L251P mutation and a BCR with autonomous signaling provide functionally equivalent but alternative oncogenic growth and survival signals in experimental systems of ABC-DLBCL. Other recurrent CARD11 mutations have weaker or virtually undetectable effects. The low prevalence of the CARD11 L251P mutation emphasizes the importance of antigen-independent autonomous BCR signaling as a frequent non-genetic immunological driver mechanism and the need for detailed dissection of the relative functional contribution of genetic and immunological drivers in order to develop optimal individualized targeted therapy for DLBCL patients.